ClpA- and ClpAP-Catalyzed Unfolding and Translocation are Differentially Coupled to ATP Binding

ClpA is an ATP-dependent chaperone essential for protein quality control in E. coli. Upon ATP binding, ClpA forms hexameric rings capable of association with the tetradecameric ClpP protease. ClpA couples ATP binding and/or hydrolysis to the unfolding and translocation of protein substrates into the central cavity of ClpP for degradation. We previously developed a single-turnover stopped-flow method sensitive to ClpA-catalyzed translocation in the absence of ClpP-catalyzed proteolysis. This method was used on unstructured substrates so that the kinetics were reflective of translocation and not unfolding. We showed that at saturating [ATP], ClpA translocated at ~20 aa s-1, with the kinetic step size, i.e., the average number of amino acids (aa) translocated between two rate-limiting steps being ~14 aa step-1. Adding ClpP increased the rate to ~36 aa s-1 and decreased the kinetic step-size to ~5 aa step-1. Here we apply this method to substrates containing folded Titin I27 domains. We report that at saturating [ATP], ClpA unfolded and translocated at ~12 aa s-1, nearly half the rate of translocation alone. However, in the presence of ClpP, ClpA exhibited a rate of ~40 aa s-1, representing no reduction in rate over translocation alone. Interestingly, unlike translocation alone, the kinetic step-size for unfolding and translocation was ~29 aa step-1 for both ClpA and ClpAP. Examining the [ATP]-dependence of the unfolding reactions revealed that the increased kinetic step-size results from the averaging of a large unfolding step-size of ~97 aa, representing cooperative unfolding of a single Titin I27 domain, followed by multiple smaller translocation steps on the newly unfolded chain. Moreover, just like translocation alone, the introduction of folds into the substrate results in different kinetics between ClpA and ClpAP. These observations further support a model where ClpP allosterically impacts ClpA-catalyzed processes.